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• W2046203977 abstract "Conventional protein kinase C (PKC) isoforms are essential serine/threonine kinases regulating many signaling networks. At cell adhesion sites, PKCα can impact the actin cytoskeleton through its influence on RhoGTPases, but the intermediate steps are not well known. One important regulator of RhoGTPase function is the multifunctional guanine nucleotide dissociation inhibitor RhoGDIα that sequesters several related RhoGTPases in an inactive form, but it may also target them through interactions with actin-associated proteins. Here, it is demonstrated that conventional PKC phosphorylates RhoGDIα on serine 34, resulting in a specific decrease in affinity for RhoA but not Rac1 or Cdc42. The mechanism of RhoGDIα phosphorylation is distinct, requiring the kinase and phosphatidylinositol 4,5-bisphosphate, consistent with recent evidence that the inositide can activate, localize, and orient PKCα in membranes. Phosphospecific antibodies reveal endogenous phosphorylation in several cell types that is sensitive to adhesion events triggered, for example, by hepatocyte growth factor. Phosphorylation is also sensitive to PKC inhibition. Together with fluorescence resonance energy transfer microscopy sensing GTP-RhoA levels, the data reveal a common pathway in cell adhesion linking two essential mediators, conventional PKC and RhoA. Conventional protein kinase C (PKC) isoforms are essential serine/threonine kinases regulating many signaling networks. At cell adhesion sites, PKCα can impact the actin cytoskeleton through its influence on RhoGTPases, but the intermediate steps are not well known. One important regulator of RhoGTPase function is the multifunctional guanine nucleotide dissociation inhibitor RhoGDIα that sequesters several related RhoGTPases in an inactive form, but it may also target them through interactions with actin-associated proteins. Here, it is demonstrated that conventional PKC phosphorylates RhoGDIα on serine 34, resulting in a specific decrease in affinity for RhoA but not Rac1 or Cdc42. The mechanism of RhoGDIα phosphorylation is distinct, requiring the kinase and phosphatidylinositol 4,5-bisphosphate, consistent with recent evidence that the inositide can activate, localize, and orient PKCα in membranes. Phosphospecific antibodies reveal endogenous phosphorylation in several cell types that is sensitive to adhesion events triggered, for example, by hepatocyte growth factor. Phosphorylation is also sensitive to PKC inhibition. Together with fluorescence resonance energy transfer microscopy sensing GTP-RhoA levels, the data reveal a common pathway in cell adhesion linking two essential mediators, conventional PKC and RhoA. IntroductionProtein kinase C (PKC) 6The abbreviations used are: PKCprotein kinase CPtdInsphosphatidylinositolPtdphosphatidylGTPγSguanosine 5′-3-O-(thio)triphosphateMDCKMadin-Darby canine kidney cellREFrat embryo fibroblastFRETfluorescence resonance energy transferGDIguanine dissociation inhibitorDLdioleinPMAphorbol 12-myristate 13-acetatesiRNAsmall interfering RNACFPcyan fluorescent proteinYFPyellow fluorescent proteinHGFhepatocyte growth factor. isoforms have a wide array of functions in cell adhesion, cytoskeleton, trafficking, and polarity (1.Ivaska J. Kermorgant S. Whelan R. Parsons M. Ng T. Parker P.J. Biochem. Soc. Trans. 2003; 31: 90-93Crossref PubMed Google Scholar, 2.Larsson C. Cell. Signal. 2006; 18: 276-284Crossref PubMed Scopus (282) Google Scholar, 3.Suzuki A. Ohno S. J. Cell Sci. 2006; 119: 979-987Crossref PubMed Scopus (570) Google Scholar) to transcription, survival, and differentiation (4.Reyland M.E. Front. Biosci. 2009; 14: 2386-2399Crossref PubMed Scopus (214) Google Scholar). Their activities can intersect with a number of signaling cascades and networks and are stimulated through several distinct receptor families. Broadly, there are three PKC subfamilies, conventional, novel, and atypical, and they operate at different subcellular sites with discrete functions (5.Mochly-Rosen D. Gordon A.S. FASEB J. 1998; 12: 35-42Crossref PubMed Scopus (509) Google Scholar, 6.Steinberg S.F. Physiol. Rev. 2008; 88: 1341-1378Crossref PubMed Scopus (625) Google Scholar). The PKCα conventional isoform may regulate a variety of functions, including redox reactions and cell survival, often upstream of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/AKT pathways and NF-κB synthesis. In addition, this isoenzyme can be localized to sites of cell adhesion and regulate actin cytoskeletal function and migration (7.Jaken S. Leach K. Klauck T. J. Cell Biol. 1989; 109: 697-704Crossref PubMed Scopus (252) Google Scholar, 8.Dovas A. Yoneda A. Couchman J.R. J. Cell Sci. 2006; 119: 2837-2846Crossref PubMed Scopus (112) Google Scholar, 9.Bass-Zubek A.E. Hobbs R.P. Amargo E.V. Garcia N.J. Hsieh S.N. Chen X. Wahl 3rd., J.K. Denning M.F. Green K.J. J. Cell Biol. 2008; 181: 605-613Crossref PubMed Scopus (113) Google Scholar). Besides the canonical pathway of PKC activation through phospholipase C-mediated generation of diacylglycerol and inositol trisphosphate, a second method of PKCα activation is known involving uncleaved phosphatidylinositol 4,5-bisphosphate (10.Corbalán-García S. García-García J. Rodríguez-Alfaro J.A. Gómez-Fernández J.C. J. Biol. Chem. 2003; 278: 4972-4980Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 11.Keum E. Kim Y. Kim J. Kwon S. Lim Y. Han I. Oh E.S. Biochem. J. 2004; 378: 1007-1014Crossref PubMed Scopus (72) Google Scholar, 12.Multhaupt H.A. Yoneda A. Whiteford J.R. Oh E.S. Lee W. Couchman J.R. J. Physiol. Pharmacol. 2009; 60: 31-38PubMed Google Scholar). Moreover, in vitro experiments reveal that Ca2+ is not required for this activation (13.Oh E.S. Woods A. Lim S.T. Theibert A.W. Couchman J.R. J. Biol. Chem. 1998; 273: 10624-10629Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) and is consistent with data showing high PtdIns(4,5)P2 affinity for the C2 domain of PKCα, mediated by a polybasic cluster of amino acids, which leads to persistence of membrane association (14.Manna D. Bhardwaj N. Vora M.S. Stahelin R.V. Lu H. Cho W. J. Biol. Chem. 2008; 283: 26047-26058Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 15.Marín-Vicente C. Nicolás F.E. Gómez-Fernández J.C. Corbalán-García S. J. Mol. Biol. 2008; 377: 1038-1052Crossref PubMed Scopus (33) Google Scholar) with decreased Ca2+ requirement (16.Guerrero-Valero M. Marín-Vicente C. Gómez-Fernández J.C. Corbalán-García S. J. Mol. Biol. 2007; 371: 608-621Crossref PubMed Scopus (52) Google Scholar).Downstream events from PKCα in fibroblast adhesion to fibronectin, for example, are not known other than an eventual up-regulation of GTP-RhoA levels, with concomitant cytoskeletal reorganization (8.Dovas A. Yoneda A. Couchman J.R. J. Cell Sci. 2006; 119: 2837-2846Crossref PubMed Scopus (112) Google Scholar, 17.Bass M.D. Morgan M.R. Roach K.A. Settleman J. Goryachev A.B. Humphries M.J. J. Cell Biol. 2008; 181: 1013-1026Crossref PubMed Scopus (95) Google Scholar). The localization of p190RhoGAP may be intrinsic to the process (17.Bass M.D. Morgan M.R. Roach K.A. Settleman J. Goryachev A.B. Humphries M.J. J. Cell Biol. 2008; 181: 1013-1026Crossref PubMed Scopus (95) Google Scholar), although a precise role for PKCα activity is unclear. The three mammalian RhoGDI isoforms are cytosolic proteins that sequester RhoGTPases in their GDP-bound form, effectively forming sinks that remove the G proteins from cycling through the guanine exchange factor and GTPase-activating protein-mediated activation/deactivation process (18.DerMardirossian C. Bokoch G.M. Trends Cell Biol. 2005; 15: 356-363Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar, 19.Dovas A. Couchman J.R. Biochem. J. 2005; 390: 1-9Crossref PubMed Scopus (312) Google Scholar). In addition, RhoGDI can interact with cytoskeletal proteins such as ezrin-radixin-moesin that could target RhoGTPase activity at a subcellular level (20.Takahashi K. Sasaki T. Mammoto A. Takaishi K. Kameyama T. Tsukita S. Takai Y. J. Biol. Chem. 1997; 272: 23371-23375Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). Therefore, it is an oversimplification to ascribe only a sequestration function to GDI proteins. Because RhoGDIα, the most abundant isoform, binds several RhoGTPases, including GDP-RhoA, -Rac1, and -Cdc42 (18.DerMardirossian C. Bokoch G.M. Trends Cell Biol. 2005; 15: 356-363Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar), regulatory mechanisms that control its function have come under some recent scrutiny (19.Dovas A. Couchman J.R. Biochem. J. 2005; 390: 1-9Crossref PubMed Scopus (312) Google Scholar, 21.DerMardirossian C. Rocklin G. Seo J.Y. Bokoch G.M. Mol. Biol. Cell. 2006; 17: 4760-4768Crossref PubMed Scopus (122) Google Scholar). RhoGDIα has two domains, an N-terminal domain of around 70 amino acids and an immunoglobulin-like C-terminal domain of ∼130 amino acids. NMR studies have shown that the N-terminal domain of the free protein is flexible in solution but with residues 9–20 and 36–58 forming transient helices (22.Golovanov A.P. Hawkins D. Barsukov I. Badii R. Bokoch G.M. Lian L.Y. Roberts G.C. Eur. J. Biochem. 2001; 268: 2253-2260Crossref PubMed Scopus (9) Google Scholar). This part of the GDI molecule is stabilized by interactions with GTPases and is therefore a potential target for regulation through protein modification, such as phosphorylation. Here, we test the hypothesis that RhoGDIα phosphorylation by PKC may represent a major step in the pathway to RhoA, with onward activation of downstream effectors regulating cell adhesion, cytoskeletal, and other cellular events.DISCUSSIONRhoGDI proteins regulate the cycling and distribution of RhoGTPases (18.DerMardirossian C. Bokoch G.M. Trends Cell Biol. 2005; 15: 356-363Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar, 19.Dovas A. Couchman J.R. Biochem. J. 2005; 390: 1-9Crossref PubMed Scopus (312) Google Scholar). Co-crystallization data of RhoGDIα bound to Cdc42 or Rac1 (38.Hoffman G.R. Nassar N. Cerione R.A. Cell. 2000; 100: 345-356Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 53.Grizot S. Fauré J. Fieschi F. Vignais P.V. Dagher M.C. Pebay-Peyroula E. Biochemistry. 2001; 40: 10007-10013Crossref PubMed Scopus (109) Google Scholar) reveal that residues 34–57 form an ordered helix-loop-helix motif characterized by hydrophobic interactions. Acquisition of negative charge at Ser-34 may disrupt the stability of the helix-loop-helix motif, interfering with the GTPase interaction and causing release of the GTPase from RhoGDI. In addition, there are extensive interactions between GDI N termini and switch I and II regions of the GTPases. GTPase binding establishes a bridge that brings regions of the N- and C-terminal domains of RhoGDI into proximity. A striking example is Arg-66 (Cdc42), the guanidinium group of which exhibits hydrogen bonding with Asp-185 as well as Pro-30 and Ala-31 of the GDI. Additionally, hydrophobic interactions between the aliphatic portion of the Arg-66 with Gln-32 and Ile-122 of the GDI were observed (38.Hoffman G.R. Nassar N. Cerione R.A. Cell. 2000; 100: 345-356Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar). Because many of the key residues of the RhoGDI N-terminal region lie immediately adjacent to Ser-34, these interactions may be disturbed by phosphorylation. However, Arg-66 of Cdc42 is highly conserved in Rac1 and RhoA, and indeed there is high sequence conservation through the switch II region, consistent with its role in GTP hydrolysis regulation, so the RhoA-specific effect of Ser-34 phosphorylation deserves further analysis.In vitro studies identifying the site and consequences of RhoGDIα phosphorylation are supported by in vivo studies with a Ser-34-phosphospecific antibody. Four widely differing cell types were investigated, and in each case endogenous phosphorylation of the Ser-34 residue could be detected. In MDCK cells, phorbol ester stimulates cytoskeletal reorganization and PKCα translocation to the membrane (43.Sciorra V.A. Daniel L.W. J. Biol. Chem. 1996; 271: 14226-14232Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), although integrin-mediated adhesion is promoted by phorbol ester in K562 cells, also associated with actin cytoskeletal reorganization (39.Lub M. van Vliet S.J. Oomen S.P. Pieters R.A. Robinson M. Figdor C.G. van Kooyk Y. Mol. Biol. Cell. 1997; 8: 719-728Crossref PubMed Scopus (46) Google Scholar). In both cell types, RhoGDIα phosphorylation on Ser-34 rapidly increased in concert with these adhesion events. Additionally, and consistent with in vitro data, RhoGDIα phosphorylation was blocked by the Gö6976 inhibitor, which has highest activity against PKCα and PKCβ1 (41.Martiny-Baron G. Kazanietz M.G. Mischak H. Blumberg P.M. Kochs G. Hug H. Marmé D. Schächtele C. J. Biol. Chem. 1993; 268: 9194-9197Abstract Full Text PDF PubMed Google Scholar) The increase in Ser-34 phosphorylation of RhoGDIα in MDCK cells responding to phorbol ester, or HGF, is particularly interesting. Both stimulants activate RhoA rather than Rac (45.Kodama A. Matozaki T. Fukuhara A. Kikyo M. Ichihashi M. Takai Y. Mol. Biol. Cell. 2000; 11: 2565-2575Crossref PubMed Scopus (106) Google Scholar, 54.Kitajo H. Shibata T. Nagayasu H. Kawano T. Hamada J. Yamashita T. Arisue M. Oncol. Res. 2003; 10: 1351-1356Google Scholar, 55.Takaishi K. Sasaki T. Kato M. Yamochi W. Kuroda S. Nakamura T. Takeichi M. Takai Y. Oncogene. 1994; 9: 273-279PubMed Google Scholar), although the molecular mechanism is unknown. The result is actin cytoskeletal reorganization and loss of cell-cell junctions. Moreover, two studies with mouse keratinocytes and KB cells showed that motility in response to these same agents was blocked both by C3 transferase and overexpression of wild-type RhoGDI (55.Takaishi K. Sasaki T. Kato M. Yamochi W. Kuroda S. Nakamura T. Takeichi M. Takai Y. Oncogene. 1994; 9: 273-279PubMed Google Scholar, 56.Nishiyama T. Sasaki T. Takaishi K. Kato M. Yaku H. Araki K. Matsuura Y. Takai Y. Mol. Cell. Biol. 1994; 14: 2447-2456Crossref PubMed Scopus (159) Google Scholar). These studies therefore implicated RhoGDI, with which the current data are entirely consistent. A key result is that introduction of the S34D mutant of RhoGDIα into MDCK cells activated the motility responses of MDCK cells without a need for PKC activation. In contrast, such responses were not seen in wild-type or S34A RhoGDI-expressing cells. However, we cannot rule out additional roles for guanine exchange factors and GTPase-activating proteins. Gentile et al. (57.Gentile A. D'Alessandro L. Lazzari L. Martinoglio B. Bertotti A. Mira A. Lanzetti L. Comoglio P.M. Medico E. Oncogene. 2008; 27: 5590-5598Crossref PubMed Scopus (27) Google Scholar) showed, for example, that HGF-promoted invasion is associated with down-regulated expression of the RacGAP, Arhgap12.Further experiments utilized the Raichu construct as a reporter for GTP-Rho levels (33.Yoshizaki H. Ohba Y. Kurokawa K. Itoh R.E. Nakamura T. Mochizuki N. Nagashima K. Matsuda M. J. Cell Biol. 2003; 162: 223-232Crossref PubMed Scopus (332) Google Scholar). This sensitive indicator combined with FRET microscopy enabled the impact of RhoGDIα phosphorylation to be assessed. Expression of the S34D form of RhoGDIα in fibroblasts yielded a FRET decline (i.e. GTP-RhoA levels increased). This provided in vivo evidence that RhoGDIα phosphorylation regulates RhoGTP levels. In contrast, no elevation in GTP-RhoA levels was seen by transfection of wild-type RhoGDIα or an S34A form. In these cases, FRET was increased, suggesting that a form of RhoGDIα that cannot be phosphorylated sequesters GDP-RhoA. Although wild-type RhoGDIα can theoretically be phosphorylated when overexpressed, increased protein levels alter the equilibrium in favor of decreased overall phosphorylation, consistent with Takaishi et al. (55.Takaishi K. Sasaki T. Kato M. Yamochi W. Kuroda S. Nakamura T. Takeichi M. Takai Y. Oncogene. 1994; 9: 273-279PubMed Google Scholar).Efficient Ser-34 phosphorylation of RhoGDIα by conventional PKC occurs in the presence of PtdIns(4,5)P2, not with the often used combination of phosphatidylserine, diacylglycerol, and calcium. This inositol phospholipid can mediate PKCα activation, and a lysine-rich-binding site in the C2 domain was identified (10.Corbalán-García S. García-García J. Rodríguez-Alfaro J.A. Gómez-Fernández J.C. J. Biol. Chem. 2003; 278: 4972-4980Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). We, and others, showed that syndecan-4-mediated activation of PKCα in the presence of PtdIns(4,5)P2 was calcium-independent (13.Oh E.S. Woods A. Lim S.T. Theibert A.W. Couchman J.R. J. Biol. Chem. 1998; 273: 10624-10629Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 35.Horowitz A. Simons M. J. Biol. Chem. 1998; 273: 25548-25551Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), and this mechanism of kinase activation corresponds well to the current data showing that the PKCα/PtdIns(4,5)P2 combination efficiently phosphorylated RhoGDIα. The C2 domain of PKCα has a higher affinity for PtdIns(4,5)P2 than phosphatidylserine, so the inositide may be a kinase-targeting site (15.Marín-Vicente C. Nicolás F.E. Gómez-Fernández J.C. Corbalán-García S. J. Mol. Biol. 2008; 377: 1038-1052Crossref PubMed Scopus (33) Google Scholar, 58.Son H. Lim Y. Kim J. Park H. Choi S. Han I. Kim W.S. Park S. Bae Y. Oh E.S. Arch. Biochem. Biophys. 2006; 454: 1-6Crossref PubMed Scopus (4) Google Scholar). Recent work shows that PKCα C2 domain may adopt a different membrane orientation when bound to PtdIns(4,5)P2 compared with phosphatidylserine (59.Landgraf K.E. Malmberg N.J. Falke J.J. Biochemistry. 2008; 47: 8301-8316Crossref PubMed Scopus (37) Google Scholar). In turn, this may modulate kinase activity, and our study provides some evidence that substrate specificity might be engendered this way. Therefore, we predict PKCα activation resulting from phospholipase C activity, i.e. the generation of diacylglycerol, would not trigger RhoGDI phosphorylation and RhoA activation. By the same token, because inositol 1,4,5-trisphosphate and Ca2+ release from the endoplasmic reticulum are consequences of phospholipase C activity, the phosphorylation of RhoGDIα is presumed to be independent of calcium fluxes. Consistent with this, we find that the PtdIns(4,5)P2-driven PKCα activation is indeed Ca2+-independent. Moreover, RhoGDIα phosphorylation persisted in the presence of a characterized phospholipase C inhibitor, U73122 (60.Horowitz L.F. Hirdes W. Suh B.C. Hilgemann D.W. Mackie K. Hille B. J. Gen. Physiol. 2005; 126: 243-262Crossref PubMed Scopus (264) Google Scholar).Our data differ from two reports suggesting that in endothelial cells Ser-96 of RhoGDIα was subject to phosphorylation by PKCα, with a downstream increase in GTP-RhoA levels (61.Knezevic N. Roy A. Timblin B. Konstantoulaki M. Sharma T. Malik A.B. Mehta D. Mol. Cell Biol. 2007; 27: 6323-6333Crossref PubMed Scopus (51) Google Scholar) or release of RhoG (62.Elfenbein A. Rhodes J.M. Meller J. Schwartz M.A. Matsuda M. Simons M. J. Cell Biol. 2009; 186: 75-83Crossref PubMed Scopus (66) Google Scholar), a close relative of Rac1 (63.van Buul J.D. Allingham M.J. Samson T. Meller J. Boulter E. García-Mata R. Burridge K. J. Cell Biol. 2007; 178: 1279-1293Crossref PubMed Scopus (168) Google Scholar). Although the methods and cells are different, we could not demonstrate phosphorylation of Ser-96 in vitro. A truncated form of RhoGDIα(67–204), missing the N-terminal region but containing Ser-96, was not phosphorylated by PKCα. Knezevic et al. (61.Knezevic N. Roy A. Timblin B. Konstantoulaki M. Sharma T. Malik A.B. Mehta D. Mol. Cell Biol. 2007; 27: 6323-6333Crossref PubMed Scopus (51) Google Scholar) utilized N-terminally green fluorescent protein-tagged RhoGDIα that may have affected the ability of PKCα to phosphorylate Ser-34. In contrast to that study, we prepared a phosphospecific antibody that revealed both the widespread occurrence of Ser-34 phosphorylation and regulation in response to PKC activation. Serine 96 lies in a consensus PKC phosphorylation site that is present in the immunoglobulin domain of mammalian RhoGDIα but not conserved across other vertebrates or isoforms. Based on structural data, the Ser-96 residue does not apparently form part of a contact site with GTPase or the hydrophobic pocket that captures the prenyl moiety. In contrast, Ser-34 is conserved across all vertebrate RhoGDIα isoforms and is present in all vertebrate β and γ isoforms for which there are data. Although Ser-34 is a major PKC phosphorylation site, in vitro experiments suggested some residual phosphorylation after GDI mutation of Ser-34 to alanine or aspartate. The amounts varied but could represent another phosphorylation site, presumably also in the N-terminal portion of RhoGDIα.Because RhoGDIα is an abundant cytoplasmic protein, its role is potentially significant. Our work complements that of DerMardirossian et al. (64.DerMardirossian C. Schnelzer A. Bokoch G.M. Mol. Cell. 2004; 15: 117-127Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar) where p21-activated kinase phosphorylation of RhoGDIα on Ser-101 and Ser-174 causes specific release of Rac1. In this case, the key sites are C-terminal and are close to each other and to the prenyl-binding cleft of the GDI (38.Hoffman G.R. Nassar N. Cerione R.A. Cell. 2000; 100: 345-356Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar). It is proposed that the negative charge may destabilize this hydrophobic interaction and cause release of the Rac1. This must be a very specific effect because Rho and Cdc42 are also prenylated yet are unaffected. A second model of RhoGDI regulation involves Tyr-156 phosphorylation by Src, but only when not complexed to a GTPase (21.DerMardirossian C. Rocklin G. Seo J.Y. Bokoch G.M. Mol. Biol. Cell. 2006; 17: 4760-4768Crossref PubMed Scopus (122) Google Scholar). This not only reduced affinity of the GDI for RhoA, Rac1, and Cdc42, but it also triggered a membrane or cortical redistribution of the protein. The current data show that Ser-34 phosphorylation can occur in free or GTPase-bound RhoGDIα. Phosphorylation on Tyr-156 was only noted in cells overexpressing a constitutively active form of Src (21.DerMardirossian C. Rocklin G. Seo J.Y. Bokoch G.M. Mol. Biol. Cell. 2006; 17: 4760-4768Crossref PubMed Scopus (122) Google Scholar), perhaps an indicator of its transient nature. Here, Ser-34 phosphorylation was observed in several different untransfected cell types, with levels changing in response to adhesion events.Focal adhesion and microfilament bundle assembly is promoted by GTP-RhoA (65.Ridley A.J. Hall A. EMBO J. 1994; 13: 2600-2610Crossref PubMed Scopus (439) Google Scholar, 66.Machesky L.M. Hall A. J. Cell Biol. 1997; 138: 913-926Crossref PubMed Scopus (304) Google Scholar), with activation of the Rho kinases, with ROCK I playing a major role in primary fibroblasts (28.Yoneda A. Multhaupt H.A. Couchman J.R. J. Cell Biol. 2005; 170: 443-453Crossref PubMed Scopus (228) Google Scholar). Rho kinases phosphorylate myosin light chain and the myosin-binding subunit of myosin phosphatase, leading to myosin II-driven contraction. The transmembrane heparan sulfate proteoglycan, syndecan-4, supports focal adhesion assembly through the binding and persistent activation of PKCα, in the presence of PtdIns(4,5)P2, an event upstream of GTP-RhoA (8.Dovas A. Yoneda A. Couchman J.R. J. Cell Sci. 2006; 119: 2837-2846Crossref PubMed Scopus (112) Google Scholar, 11.Keum E. Kim Y. Kim J. Kwon S. Lim Y. Han I. Oh E.S. Biochem. J. 2004; 378: 1007-1014Crossref PubMed Scopus (72) Google Scholar, 25.Lim S.T. Longley R.L. Couchman J.R. Woods A. J. Biol. Chem. 2003; 278: 13795-13802Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). In MDCK cells, Rho kinase activity is required both for focal adhesion/stress fiber formation but also lamellipodial ruffle stabilization, possibly through phosphorylation of adducin (29.Fukata Y. Kimura K. Oshiro N. Saya H. Matsuura Y. Kaibuchi K. J. Cell Biol. 1998; 141: 409-418Crossref PubMed Scopus (181) Google Scholar, 67.Royal I. Lamarche-Vane N. Lamorte L. Kaibuchi K. Park M. Mol. Biol. Cell. 2000; 11: 1709-1725Crossref PubMed Scopus (242) Google Scholar). However, the upstream events that lead to the accumulation and targeting of GTP-RhoA were not known. Here, evidence suggests that a major downstream target of PKCα/PtdIns(4,5)P2 is RhoGDIα with phosphorylation on Ser-34. The result of phosphorylation is release or decreased capture of the GTPase, leading to nucleotide exchange and interaction with downstream effectors. IntroductionProtein kinase C (PKC) 6The abbreviations used are: PKCprotein kinase CPtdInsphosphatidylinositolPtdphosphatidylGTPγSguanosine 5′-3-O-(thio)triphosphateMDCKMadin-Darby canine kidney cellREFrat embryo fibroblastFRETfluorescence resonance energy transferGDIguanine dissociation inhibitorDLdioleinPMAphorbol 12-myristate 13-acetatesiRNAsmall interfering RNACFPcyan fluorescent proteinYFPyellow fluorescent proteinHGFhepatocyte growth factor. isoforms have a wide array of functions in cell adhesion, cytoskeleton, trafficking, and polarity (1.Ivaska J. Kermorgant S. Whelan R. Parsons M. Ng T. Parker P.J. Biochem. Soc. Trans. 2003; 31: 90-93Crossref PubMed Google Scholar, 2.Larsson C. Cell. Signal. 2006; 18: 276-284Crossref PubMed Scopus (282) Google Scholar, 3.Suzuki A. Ohno S. J. Cell Sci. 2006; 119: 979-987Crossref PubMed Scopus (570) Google Scholar) to transcription, survival, and differentiation (4.Reyland M.E. Front. Biosci. 2009; 14: 2386-2399Crossref PubMed Scopus (214) Google Scholar). Their activities can intersect with a number of signaling cascades and networks and are stimulated through several distinct receptor families. Broadly, there are three PKC subfamilies, conventional, novel, and atypical, and they operate at different subcellular sites with discrete functions (5.Mochly-Rosen D. Gordon A.S. FASEB J. 1998; 12: 35-42Crossref PubMed Scopus (509) Google Scholar, 6.Steinberg S.F. Physiol. Rev. 2008; 88: 1341-1378Crossref PubMed Scopus (625) Google Scholar). The PKCα conventional isoform may regulate a variety of functions, including redox reactions and cell survival, often upstream of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/AKT pathways and NF-κB synthesis. In addition, this isoenzyme can be localized to sites of cell adhesion and regulate actin cytoskeletal function and migration (7.Jaken S. Leach K. Klauck T. J. Cell Biol. 1989; 109: 697-704Crossref PubMed Scopus (252) Google Scholar, 8.Dovas A. Yoneda A. Couchman J.R. J. Cell Sci. 2006; 119: 2837-2846Crossref PubMed Scopus (112) Google Scholar, 9.Bass-Zubek A.E. Hobbs R.P. Amargo E.V. Garcia N.J. Hsieh S.N. Chen X. Wahl 3rd., J.K. Denning M.F. Green K.J. J. Cell Biol. 2008; 181: 605-613Crossref PubMed Scopus (113) Google Scholar). Besides the canonical pathway of PKC activation through phospholipase C-mediated generation of diacylglycerol and inositol trisphosphate, a second method of PKCα activation is known involving uncleaved phosphatidylinositol 4,5-bisphosphate (10.Corbalán-García S. García-García J. Rodríguez-Alfaro J.A. Gómez-Fernández J.C. J. Biol. Chem. 2003; 278: 4972-4980Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 11.Keum E. Kim Y. Kim J. Kwon S. Lim Y. Han I. Oh E.S. Biochem. J. 2004; 378: 1007-1014Crossref PubMed Scopus (72) Google Scholar, 12.Multhaupt H.A. Yoneda A. Whiteford J.R. Oh E.S. Lee W. Couchman J.R. J. Physiol. Pharmacol. 2009; 60: 31-38PubMed Google Scholar). Moreover, in vitro experiments reveal that Ca2+ is not required for this activation (13.Oh E.S. Woods A. Lim S.T. Theibert A.W. Couchman J.R. J. Biol. Chem. 1998; 273: 10624-10629Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) and is consistent with data showing high PtdIns(4,5)P2 affinity for the C2 domain of PKCα, mediated by a polybasic cluster of amino acids, which leads to persistence of membrane association (14.Manna D. Bhardwaj N. Vora M.S. Stahelin R.V. Lu H. Cho W. J. Biol. Chem. 2008; 283: 26047-26058Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 15.Marín-Vicente C. Nicolás F.E. Gómez-Fernández J.C. Corbalán-García S. J. Mol. Biol. 2008; 377: 1038-1052Crossref PubMed Scopus (33) Google Scholar) with decreased Ca2+ requirement (16.Guerrero-Valero M. Marín-Vicente C. Gómez-Fernández J.C. Corbalán-García S. J. Mol. Biol. 2007; 371: 608-621Crossref PubMed Scopus (52) Google Scholar).Downstream events from PKCα in fibroblast adhesion to fibronectin, for example, are not known other than an eventual up-regulation of GTP-RhoA levels, with concomitant cytoskeletal reorganization (8.Dovas A. Yoneda A. Couchman J.R. J. Cell Sci. 2006; 119: 2837-2846Crossref PubMed Scopus (112) Google Scholar, 17.Bass M.D. Morgan M.R. Roach K.A. Settleman J. Goryachev A.B. Humphries M.J. J. Cell Biol. 2008; 181: 1013-1026Crossref PubMed Scopus (95) Google Scholar). The localization of p190RhoGAP may be intrinsic to the process (17.Bass M.D. Morgan M.R. Roach K.A. Settleman J. Goryachev A.B. Humphries M.J. J. Cell Biol. 2008; 181: 1013-1026Crossref PubMed Scopus (95) Google Scholar), although a precise role for PKCα activity is unclear. The three mammalian RhoGDI isoforms are cytosolic proteins that sequester RhoGTPases in their GDP-bound form, effectively forming sinks that remove the G proteins from cycling through the guanine exchange factor and GTPase-activating protein-mediated activation/deactivation process (18.DerMardirossian C. Bokoch G.M. Trends Cell Biol. 2005; 15: 356-363Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar," @default.
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